Integrins are a family of cell adhesion/signaling molecules that play an important role in development, wound repair, angiogenesis, immunity, and tissue integrity. Regulation of specific integrins is a potentially powerful therapeutic target for diverse diseases including cancer and autoimmunity. A remarkable characteristic of integrins is the degree of cellular regulation that can be achieved without altering protein expression levels. For example, the integrin Lymphocyte Function Associated-1 (LFA-1) is initially inactive on resting lymphocytes, but adhesion activity is rapidly increased by exposure to chemokines or antigen. Changes in LFA-1 affinity for ligand are important, but are not the exclusive mechanism for regulation; there is mounting evidence that integrin interactions with the cytoskeleton are equally important. Our hypothesis is that integrin activity is regulated by a multistep cascade with the following major steps: 1) intermediate affinity integrin is initially attached to the cytoskeleton to prevent interaction with ligands, 2) activation releases the intermediate affinity integrin from cytoskeletal constraints to increase ligand binding, 3) ligand binding induces a conformational change in the integrin to the high affinity conformation, and 4) the ligated integrin binds cytoplasmic factors that regulate local mechanical properties to enhance two dimensional affinity of integrin-ligand interaction. We will test these hypotheses by examining the physiological chemistry of integrin interactions (bonds) using ligands in supported planar bilayers to determine the kinetic and equilibrium binding of LFA-1 under different conditions. We will also make use of single molecule measurements where appropriate. In Aim 1 we will compare differentiation, chemokine and antigen receptor mediated LFA-1 activation. In Aim 2 we will determine how different modes of lymphocyte activation affect lateral mobility of different forms of LFA-1 as an assay for release from the cytoskeleton. In Aim 3 we will determine how defects in the cytoskeleton regulators Wiskott Aldrich Syndrome protein (WASP) and WASP interacting protein (WIP) influence the lateral mobility of LFA-1 and its ability to mediate regulated adhesion. In Aim 4 we will determine the effect of signaling pathways including those mediated by Adhesion and Degranulation promoting Adapter Protein (ADAP) and Protein Kinase C-0 (PKC-0) on LFA-1 mobility and clustering. These experiments should provide new insight into regulation of integrin function and identify potential therapeutic targets for regulation of leukocyte integrins.